8 well glass bottom chamber slides

After blood collection, the aorta was clamped above its entry into the heart, and a 5 mM EDTA solution was injected slowly into the right and left ventricles to clear the coronary vessels. The heart was removed and the aorta was mounted onto a perfusion apparatus (Radnoti LLC, Covina, CA) for retrograde perfusion with a modified Ca²⁺-free Krebs-Henseleit buffer (KHB) containing collagenase (0.18 g/50 ml KHB; Worthington Biochemical Corp., Lakewood, NJ). Enzymatic digestion occurred at 37°C for ~30 min. and then terminated with EDTA-containing KHB. The atria and right ventricle were removed, and the left ventricle was gently teased apart to isolate cardiomyocytes. Calcium was reintroduced to the isolated myocytes over a 20 min. period, after which the myocytes were either maintained in Tyrode’s buffer for immediate IonOptix analysis or plated onto laminin-coated coverslips or 8-well glass-bottom chamber slides (Ibidi, GmbH) in medium M199 containing 10% FBS and 2,3-butanedione monoxime (BDM, 10 mM). Myocytes were allowed to adhere for 1 to 2 hours prior to staining or fixation for microscopy.

U2OS were plated on 8-well glass-bottom chamber slides (Ibidi). Imaging of TDP-43^{∆NLS-clover} were done using the 20x objective (0.8. air. 0.55 mm) on Zeiss LSM880 confocal microscope with settings: scan resolution = 1024 × 1024; scan speed level = 8; pinhole = 20; Gain was set at 500 and offset was set to 600 to allow all the signal in the picture is in linear range. 488 nm were used for excitation and the signals from 500 nm to 600 nm were collected. Mean intensity of TDP-43^{∆NLS-clover} in the cytoplasm was measured using FIJI (ImageJ) by circling the area of the cells with fluorescence. Dot plotting of the intensity from 23 cells with TDP-43 granules, and 27 cells without granules from nine images.